Analytical Matrix Effect Research Articles

Pharmacokinetic and tissue distribution analysis of bioactive compounds from Fuke Qianjin capsules in rats by a validated UPLC[sbnd]MS/MS method

Fuke Qianjin capsules (FKQJ) exhibit obvious advantages and characteristics in the treatment of pelvic inflammatory disease. At present, information regarding the in vivo process of FKQJ is lacking, which has become a bottleneck in further determining the therapeutic effect of this traditional Chinese medicine. In the present study, a sensitive, simple and reliable method was developed and validated for the simultaneous quantification of 12 main components (4 flavonoids, 4 alkaloids, 2 phthalides and 2 diterpene lactones) in plasma and seven tissues of rats to study the pharmacokinetic and distribution characteristics of these components in vivo by using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the first time. Plasma and tissue were prepared by protein precipitation with acetonitrile and methanol, followed by its separation on a Waters Acquity UPLC BEH C18 column. The quantification was performed via multiple reaction monitoring (MRM) by a triple quadrupole mass spectrometer under positive electrospray ionization (ESI) mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect and stability. For 12 analytes, the low limit of quantification (LLOQs) reached 0.005–2.44 ng/mL, and all calibration curves showed good linearity (r2 ≥ 0.990) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.96%, and the accuracies were in the range of 85.29%−114.97%. Extraction recoveries and matrix effects of analytes were acceptable. The pharmacokinetic results showed that the main components could be absorbed quickly, had a short residence time, and were eliminated quickly in vivo. At different time points, the 12 components were widely distributed with uneven characteristics in the body, which tended to be distributed in the liver, kidney and lung and to a lesser extent in the uterus, brain and heart. The pharmacokinetic process and tissue distribution characteristics of FKQJ were expounded in this study, which can provide a scientific theory for in-depth development of FKQJ and guide FKQJ use in the clinic.

Simultaneous determination of six triterpenoid saponins in beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study after oral administration of the extract of the Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves

AbstractA rapid and simple ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous determination of six analytes from the Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves (ESL) in beagle dog plasma for the first time, including 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside-29-hydroxy oleanolic acid, 3-O-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranoside-29-hydroxy oleanolic acid, 3-O-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranosyl-30-norlean-12,20 (29) –dien-28-olic acid, ciwujianoside E, guaianin N, and eleutheroside K. The chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using a gradient elution way with a mobile phase of acetonitrile-water containing 0.1% formic acid. Analytes were detected on a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source with multiple reaction monitoring (MRM) mode. Calibration curves were all linear (r ≥ 0.9933) over the concentration range. The mean extraction recoveries and matrix effect of analytes and I.S. were ranged from 80.26% to 98.32% and from 91.27% to 111.67%, respectively. The intra-day and inter-day precision were ranged from 2.20% to 14.81%, and the accuracy range was 1.60–14.60%. The analytical method was successfully applied for the pharmacokinetic characteristics of the six analytes in beagle plasma after oral administration of ESL extracts. The T1/2 of six analytes was more than 3.09 ± 0.78 h.

Simultaneous determination of multiple constituents of Qi-Lin pill by UPLC-MS/MS: Applications to pharmacokinetics and testicular tissue distribution in rats

Qi-Lin pill (QLP) is an effective traditional Chinese medicine prescription (TCMP) that has been used for the treatment of the oligoasthenozoospermia in China. Recently, some articles described the pharmacological effects of QLP and multiple ingredients in QLP contribute to its effects. However, the pharmacokinetic and target tissue distribution data of QLP are still unknown. In the present study, according to the Bioanalytical Method Validation Guidance of FDA, a sensitive and selective UPLC-MS/MS method was developed and validated for simultaneous determination of multiple constituents in rat plasma and testicular tissue, including morusimic acid A, codonopyrridium B, magnoflorine, emodin, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), ecliptasaponin A, paeoniflorin, albiflorin, gallic acid, danshensu, salvianolic acid A, catechin, isosinensetin, nobiletin, formononetin, calycosin, icariside II, icariin and epimedin C. For 19 analytes, the LLOQs reached 0.01–4 ng/mL. And all calibration curves showed favorable linearity (r ≥ 0.9903) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.92 %, and the accuracies (as relative error) were in the range of − 6.44 % to 6.22 %. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The method was successfully applied for the pharmacokinetics and testis distribution of multiple chemical constituents in QLP after a single oral dose. As a result, high exposure of danshensu, gallic acid, paeoniflorin and albiflorin were observed in rat plasma and testicular tissue. Among the flavonoids, isosinensetin and nobiletin had high exposure in testicular tissue. Moreover, alleviation of progesterone reduction was evaluated in H2O2-induced R2C leydig cells, and danshensu, gallic acid, paeoniflorin, albiflorin and nobiletin showed potent activity. Therefore, these five components were considered to be the effective components of QLP due to their relatively high exposure in vivo and biological activity. This finding also provided relevant information on action mechanism of QLP in the treatment of oligoasthenozoospermia.

Rapid and sensitive analytical strategy for multi-class antibiotic residues analysis in aquatic products with amphiphilic magnetic polymer particles as an effective cleanup adsorbent

Multi-class antibiotic analysis in aquatic products is still challenging owing to the complex matrix and low co-extraction efficiency of various antibiotics. A facile and sensitive sample pretreatment method based on magnetic dispersive solid-phase extraction was thus developed via systematic optimization of extraction and purification procedure. The multi-class antibiotics can simultaneously be extracted by acetonitrile-2 % acetic acid. Amphiphilic magnetic particles and C18 were used as adsorbents to remove matrix interferences, affording significantly reduced matrix effects of analytes. Under the optimum conditions, satisfactory linearity, recovery, precision, and sensitivity were achieved. The method limits of quantification were 0.25–0.5 μg kg−1. Besides, it displayed obvious advantages in operation convenience and efficiency due to the usage of magnetic particles. The developed method was successfully used to analyze antibiotic residues in both freshwater and seafood products, manifesting its suitability for antibiotic residues analysis in aquatic products.

An LC-MS/MS Method for the Pharmacokinetic and In Vitro Metabolism Studies of Praeruptorin A in Rat

Objective: The study aims to investigate the pharmacokinetic profile of Praeruptorin A and khellactone and in vitro hydrolysis of praeruptorin A to khellactone in different biological samples. Methods: A LC-MS/MS method was established. Analytes and internal standard (IS) were isolated using the protein precipitation method and then separated on a Thermo BDS Hypersil C18 (2.1 mm×50 mm, 2.4μm) column using a mobile phase consisting of 0.05% formic acid solution and acetonitrile. Samples were analyzed in positive electrospray-ionization (ESI) mode using multiple reaction monitoring (MRM). Results: The calibration plots gave desirable linearity (r2>0.99) in the concentration range from 0.99-990.0 and 2.0-2000.0 ng/mL for Praeruptorin A and khellactone, respectively. In addition, the LOQs of these analytes were sufficient for vivo pharmacokinetic study and vitro hydrolysis study of Praeruptorin A. The intra-batch and inter-batch precision were all within 14.05%, and the accuracy was between 89.39% and 109.50%. The extraction efficiency of PA and khellactone ranged from 76.35 ~ 89.58%. The matrix effects of analytes and the IS were between 89.67% ~ 105.26%. Conclusion: The liver CYPs mediated by the metabolism of PA may contribute to the systemic exposure of its active metabolite, khellactone, in rats.

LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study.

A rapid and sensitive ultra-performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol-mediated precipitation. Chromatographic separation was achieved within 3min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1mm, i.d. 1.7 μm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1-1,000 ng/mL, with the lower limit of quantification of 1ng/mL. The intra- and inter-day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax , 0.23-0.32 h) and reached the maximum concentration of 0.82-1.75 μg/mL after oral administration. Both seneciphylline and seneciphylline N-oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43-10.31%) was related to the extensive metabolism in the liver and microflora.

Effect of the Mobile Phase Compositions on the Confirmation Analysis of Some Prohibited Substances in Sport by LC–ESI–MS/MS

In this study, the influence of the different mobile phase compositions on the sensitivity and chromatographic performance of prohibited small peptides (< 2 kDa), their metabolites, and non-peptide substances (anamorelin, ibutamoren) were demonstrated using LC–electrospray ionization coupled with triple quadrupole tandem mass spectrometry (LC–ESI–MS/MS). In the mobile phase, widely used mobile phase additives, ammonium formate (AF), formic acid (FA), and dimethylsulfoxide (DMSO) were employed with different organic phase modifiers such as acetonitrile (ACN) and methanol (MeOH). To evaluate the limit of detection (LOD) and matrix effect of analytes, urine samples were prepared by solid-phase extraction (SPE), and then, their analysis was performed by LC–ESI–MS/MS. LOD values of analytes were found to be between 0.05–2 ng mL−1 except for ibutamoren, anamorelin, and leuprolide (5–9), which had LOD values of lower than 0.05 ng mL−1. The best results related to sensitivity and chromatographic performance for the analysis of the analytes were achieved using ACN-based mobile phase containing 0.1% FA, which led to less ion suppression for most analytes compared to other employed mobile phase compositions. The proposed method to use for confirmation analysis of analytes was validated with respect to selectivity, carryover, precision, the limit of identification (LOI), recovery, matrix effect, and robustness. The applicability of the method in real urine samples was demonstrated by analyzing two different excretion urine samples containing leuprolide and triptorelin.

Pharmacokinetics and tissue distribution study of 15 ingredients of Polygonum chinense Linn extract in rats by UHPLC-MS/MS.

A rapid and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of 15 bioactive ingredients in rat plasma and tissues after oral administration of Polygonum chinense Linn extract (PCE). After addition of internal standards (ISs; rutin and danshensu), plasma and tissue samples were pre-treated by protein precipitation with acetonitrile-ethanol. The chromatographic separation was performed on an Agilent ZORBAX RRHD Eclipse Plus C18 column with gradient elution using a mobile phase composed of methanol and water (containing 0.2% acetic acid) at a flow rate of 0.3 mL min-1 . Mass spectrometric detection was carried out using a mass spectrometer in both positive and negative ion electrospray ionization modes by multiple reaction monitoring. The method provided excellent linearity, and the lower limit of quantification range 0.5-30 ng mL-1 for all analytes. The intra- and inter-day precision were less than 9.12% and the accuracy ranged from -4.02% to 6.32%, respectively. The mean extraction recovery and matrix effect of analytes and ISs ranged from 83.65% to 109.20%. The method was successfully applied to the pharmacokinetics and tissue distribution study of 15 ingredients of PCE in rats.

Isotope-labeled internal standards and grouping scheme for determination of neonicotinoid insecticides and their metabolites in fruits, vegetables and cereals – A compensation of matrix effects

Neonicotinoid insecticides (NIs) are widely used. However they produce high levels of residues and are toxic to non-target organisms, especially when their metabolites exhibit comparable or elevated toxicities. In this study, we established a multi-residue analytical method for analyzing 20 compounds – 8 NIs and their 11 major toxic metabolites. QuEChERS pretreatment and ultra-high-performance liquid chromatography–tandem mass spectrometry were used to detect residues in fruits, vegetables and cereals. Determination time was shortened to 6 min. Average recoveries of all compounds were in the range of 90.1–105.5%, with relative standard deviations lower than 15.0% and the limit of quantitation being 10 µg kg−1. For the first time, 8 NIs and 11 major toxic metabolites were grouped simultaneously, such that the matrix effect of analytes was satisfactorily corrected using their own isotopically labeled molecules; other NIs and metabolites in the same group were also corrected. This is the first report that simultaneously quantified multiple metabolites of eight NIs in multiple matrices.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.

Determination of the phytochemical composition of Jingning fang and the in vivo pharmacokinetics of its metabolites in rat plasma by UPLC-MS/MS.

Jingning fang (JNF) is an effective Traditional Chinese Medicine (TCM) which is used for the treatment of Attention Deficit Hyperactivity Disorder (ADHD). To clarify the bioactive constituents of JNF, a Thermo Q Exactive™ Plus Orbitrap™ mass spectrometer was used in this study. More than 127 chemical compounds were isolated and identified tentatively in the JNF extract, while 42 prototype constituents with 4 potential metabolites were identified tentatively in rat plasma. A method for simultaneous determination of polygalaxanthone III (PAIII), sibiricose A5 (A5), sibiricose A6 (A6), 3, 6'-disinapoyl sucrose (3,6'-DISS), tenuifoliside C (TEC), tenuifolin B (TNB), verbascoside (VCE), heterophyllin B (HEB) and schisandrin (SCH) in rat was developed and validated using polydatin (PLN) and psoralen (PSN) as internal standards. All calibration curves proved favorable linearity (R2≥0.9923) in linear ranges. The lower limit of quantification (LLOQ) was 2.5ng/mL for PAIII, A5, 3, 6'-DISS, TNB, VCE, HEB and SCH, 1.0ng/mL for A6 and TEC, respectively. Intra-day and inter-day precisions didn't exceed 14.0% for all the analytes. Extraction recoveries and matrix effects of analytes and IS were acceptable. The validated method has been successfully applied to the pharmacokinetics (PK) studies of the nine compounds in JNF. These findings are useful for predicting the bioactive components of JNF, and will aid in optimizing dose regimens of the drug.

Development of a UPLC-MS/MS Method for Simultaneous Determination of Six Flavonoids in Rat Plasma after Administration of Maydis stigma Extract and Its Application to a Comparative Pharmacokinetic Study in Normal and Diabetic Rats.

Maydis stigma is an important medicine herb used in many parts of the world for treatment of diabetes mellitus, which main bioactive ingredients are flavonoids. This paper describes for the first time a study on the comparative pharmacokinetics of six active flavonoid ingredients of Maydis stigma in normal and diabetic rats orally administrated with the decoction. Therefore, an efficient and sensitive ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of six anti-diabetic ingredients (cynaroside, quercetin, luteolin, isorhamnetin, rutin and formononetin) of Maydis stigma in rat plasma has been developed and validated in plasma samples, which showed good linearity over a wide concentration range (r2 > 0.99), and gave a lower limit of quantification of 1.0 ng·mL−1 for the analytes. The intra- and interday assay variability was less than 15% for all analytes. The mean extraction recoveries and matrix effect of analytes and IS from rats plasma were all more than 85.0%. The stability results showed the measured concentration for six analytes at three QC levels deviated within 15.0%. The results indicated that significant differences in the pharmacokinetic parameters of the analytes were observed between the two groups of animals, whereby the absorptions of these analytes in the diabetic group were all significantly higher than those in the normal group, which provides an experimental basis for the role of Maydis stigma in anti-diabetic treatment.

A simple and rapid analytical method based on solid-phase extraction and liquid chromatography–tandem mass spectrometry for the simultaneous determination of free catecholamines and metanephrines in urine and its application to routine clinical analysis

ObjectivesUrinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting. MethodsFollowing SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines. ResultsThe performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163μg/L, 7.4–2,359μg/L, 5.4–2,825μg/L, 3.5–2,466μg/L, and 3.7–2,569μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines. ConclusionsThe facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

A comprehensive LC/MS assay for biogenic amines in urine

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

Laser-induced breakdown spectroscopy for quantitative spectrochemical analysis of geological materials: Effects of the matrix and simultaneous determination

A microscopic laser-induced breakdown spectrometer was used to evaluate the analytical matrix effect commonly observed in the analysis of geological materials. Samples were analyzed in either the powder or pressed pellet forms. Calibration curves of a number of iron and aluminum compounds showed a linear relationship between the elemental concentration and peak intensity. A direct determination of elemental content can thus be made from extrapolation on these calibration curves. To investigate matrix effects, synthetic model samples were prepared from various iron and aluminum compounds spiked with SiO 2 and CaCO 3. The addition of these matrices had a pronounced analytical effect on those compounds prepared as pressed pellets. However, results indicated the absence of matrix effects when the samples were presented to the laser as loose powders on tape and results were compared to certified values, indicating the reliability of this approach for accurate analysis, provided the sample particle diameters are greater than ≈100 μm. Finally, the simultaneous analysis of two different elements was demonstrated using powders on tape.

Attomole Electrophoretic Analysis of Catecholamines Using Copper-Catalyzed Intramolecular Cyclization

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

Induction of the acute phase reaction increases heparin-binding proteins in rat plasma

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.